Kalamycinic acid and derivatives and their production



United States Patent 3,524,868 KALAMYCINIC ACID AND DERIVATIVES AND THEIR PRODUCTION Herman Hoeksema, Cooper Township, Kalamazoo County, Mich, assignor to The Upjohn Company, Kalamazoo, Mich., a corporation of Delaware No Drawing. Continuation-impart of application Ser. No. 677,038, Oct. 23, 1967. This application Nov. 9, 1967, Ser. No. 681,883

Int. Cl. C07d 7/20 US. Cl. 260-6452 14 Claims ABSTRACT OF Tm DISCLOSURE Novel alkaline degradation product of the antibiotic kalamycin named kalamycinic acid, and alkyl and acyl derivatives thereof; dehydrokalamycinic acid and alkyl and acyl derivatives thereof; and 4-deoxy-4-halo derivatives of esters of kalamycinic acid, and processes for preparing the same. Also, a process for converting kalamycinic acid to kalamycin. The novel compounds of this invention have antifungal activity and can be used as antifungal agents.

CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of pending application Ser. No. 677,038, filed Oct. 23, 1967, now abandoned.

BRIEF SUMMARY OF THE INVENTION This invention is directed to novel compounds derived from the antibiotic kalamycin and to processes for producing the same.

Kalamycin is an antibiotic obtainable as an elaboration product of a kalamycin-producing actinomycete. Methods for the production, recovery and purification of lralamycin are described in U.S. 3,300,3 82. Kalamycin is particularly suitable as an antifungal agent, though it is also active against various Gram-positive and Gram-negative bacteria. The structure of kalamycin is now determined and can be shown to be as follows:

It has now been found, according to this invention, that kalamycin can be converted to novel compounds. More particularly, the invention relates to kalamycinic acid, 3,4,5,10-tetrahydro-4,9-dihydroxy-l-methyl-S,10 dioxolH-naphtho[2,3-c]pyran-3-acetic acid, a compound having the following formula:

Ha I O OH II its salts, and to its preparation.

3,524,868 Patented Aug. 18, 1970 Still more particularly, the invention is directed to esters of kalamycinic acid having the formula:

HOI (H) 0 Ha wherein R is alkyl of from 1 to 16 carbon atoms, inclusive, and the isomeric forms thereof, their salts, and to their preparation.

Still more particularly, the invention relates to 4,9-di- O-alkyl and 4,9-di-O-acyl derivatives of compound III having the formula:

kalamycinic acid and derivatives thereof having the formula:

wherein R is hydrogen or alkyl of from 1 to 16 carbon atoms, inclusive, and isomeric forms thereof; R is hydrogen, or alkyl of from 1 to 16 carbon atoms, inclusive, and isomeric forms thereof, or hydrocarbon carboxylic acid acyl of from 2 to 18 carbon atoms, inclusive; halo-,

nitro-, hydroXy-, amino-, cyano-, thiocyano, and loweralkoxy-substituted hydrocarbon carboxylic acid acyl of from 2 to 18 carbon atoms, inclusive; and lower-alkoxycarbonyl, and to their preparation.

Still more particularly, the invention relates to 4-deoxy- 4-halo derivatives of esters of kalamycinic acid having the formula:

RO O CH3 0 X VI wherein R is alkyl of from 1 to 16 carbon atoms, inclusive, and isomeric forms thereof; R is hydrogen or hydrocarbon caboxylic acid acyl of from 2 to 18 carbon atoms, inclusive; halo-, nitro-, hydroXy-, amino-, cyano-, thiocyano-, and loWeralkoxy-substituted hydrocarbon carboxylic acid acyl of from 2 to 18 carbon atoms, inclusive, and loweralkoxycarbonyl; X is halo, and to their preparation.

Still more particularly, the invention relates to a process for the conversion of kalamycinic acid to kalamycin.

3 DETAILED DESCRIPTION OF THE INVENTION Kalamycinic acid (II) is obtained by alkaline hydrolysis of kalamycin with an alkali metal or alkaline earth metal base or ammonium hydroxide. For example, upon reacting kalamycin with 1 N sodium hydroxide there is produced kalamycinic acid. The reaction is stopped by neutralizing the reaction mixture with an inorganic acid,

for example, hydrochloric acid. The alkalinehydrolysis can be carried out with a base, as disclosed above, varying in strength from 0.1 N to 6 N. The time of the reaction will vary between minutes to 10 hours depending upon the strength of the base used. Also, the temperature of the reaction can be within a range of 0 to C. (room emperature is preferred).

Kalamycinic acid is recovered from the neutralized reaction mixture by extraction with a solvent in which kalamycinic acid is soluble, for example, chloroform. The chloroform extract can be washed with water, dried over sodium sulfate, and evaporated to dryness. Crystalline kalamycinic acid can be obtained from this product by dissolving it in ethyl acetate, drying the solution over sodium sulfate and evaporating to a small volume to induce crystallization of kalamycinic acid.

Kalamycinic acid can be converted to kalamycin by reacting kalamycinic acid with a lower alcohol in the present of a volatile inorganic acid, for example, HCl, HBr, and the like. Suitable alcohols are methanol, ethanol, propanol, butanol, and isomeric forms thereof. The temperature of the reaction can be within a range of 0 C. to 25 C. This process is useful in a kalamycin recovery process when kalamycin is degraded to kalamycinic acid.

Kalamycinic acid esters can be prepared by reacting kalamycin (I) with alcohol in the presence of an inorganic acid. For example, treatment of kalamycin with ethanolic hydrogen chloride at room temperature affords an aquilibrium mixture of kalamycin and ethyl kalamycinate (III-wherein R is ethyl). Alcohols of from 1 to 16 carbon atoms can be used in the reaction, for example, methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decaol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, and hexadecanol. Inorganic acids which can be used in the process can be of the type represented by HCl, HBr, H 80 H PO and similar acids. The time to attain equilibrium varies with temperature. The temperature can be varied betwn 0- C. In this range, the time will vary between about 45 minutes and 6 hours. For example, at room temperature the equilibrium generally is obtained at about 90 minutes. The course of the reaction can be followed by the use of silica gel chromatography using a solvent system consisting of ethyl acetate-cyclohexane to develop the column. The reaction can be stopped either by rapid evaporation of the alcoholic hydrogen chloride, in vacuo, or by carefully neutralizing the reaction mixture to about pH 6 with a mineral base, for example, sodium hydroxide.

Upon permitting the above reaction to continue, the kalamycinic acid ester reacts further to produce two products, viz., an ester of dehydrokalamycinic acid having the structural Formula Vwherein R is hydrogen, and, an ester of 4-deoxy-4-halo kalamycinic acid, VI-Wherein R is hydrogen.

Compound V (wherein R is hydrogen) predominates under more stringent reaction conditions, i.e., longer time and/or use of refluxing temperatures. The recovery of Compound V from the reaction mixture can be accomplished by evaporating the reaction mixture to dryness. The product thus obtained can be chromatographed on a silica gel COlIJmn developed with ethyl acetate-cyclohexane from which fractions can be collected, evaporated to dryness, and then the ester of dehydrokalamycinic acid crystallized from hot Skellysolve B (isomeric hexanes).

Esters of dehydrokalamycinic acid can be converted to with a base, for example, NaOH, Na CO and the like, at a pH of about 10-12.

The second product (VI-wherein R is hydrogen) of the continued reaction of kalamycin with an alcohol in the presence of a hydrohalic acid, predominates under milder reaction conditions. For example, upon reacting kalamycin with ethanolic hydrogen chloride at room temperature for 3 /2 hours there is obtained ethyl 4-deoxy-4- chlorokalamycinate. The reaction can be stopped by rapid evaporation of the alcoholic hydrogen chloride in vacuo, or by carefully neutralizing the reaction mixture to about pH 6 with a mineral base, for example, sodium hydroxide. The course of the reaction can be followed by thinlayer chromatography using silica gel developed with ethyl acetate-cyclohexane (3:5). Upon reacting kalamycin with ethanolic hydrogen bromide at room temperature for 3 /2 hoursthere is obtained ethyl 4-deoxy-4-bromokalamycinate. Thus, by using a different hydrohalic acid in the above reaction, it is possible to substitute a different halo group on the 4-position of the molecule.

Kalamycinic acid (II), esters of kalamycinic acid (III), dehydrokalamycinic acid (V-wherein R and R are hydrogen), and esters of dehydrokalamycinic acid (V wherein R is hydrogen) can be alkylated or acylated at the hydroxyl occurring on the 9 or 4 position of the molecule, whichever is the case. For example, kalamycinic acid (II) and esters of kalamycinic acid (III) can be alkylated or acylated on the 9 and 4 positions of the molecule to produce 4,9-di-O-alkyl derivatives of kalamycinic acid and esters of kalamycinic acid (IV-wherein R is alkyl) or, 4,9-di-O-acyl derivates of kalamycinic acid and esters of kalamycinic acid (IVwherein R is acyl). Dehydrokalamycinic acid and esters of dehydrokalamycinic acid (Vwherein R is hydrogen) can be be alkylated or acylated to produce 9-O-alkyl derivatives of dehydrokalamycinic acid and the esters of dehydrokalamycinic acid (Vwherein R is alkyl), or 9-O-acyl derivatives of dehydrokalamycinic acid and the esters of dehydrokalamycinic acid (Vwherein R in acyl). 4-deoxy-4-halo derivatives of esters of kalamycinic acid (VI wherein R is hydrogen) can be acylated to produce 9-O- acyl derivatives (VIwherein R is acyl).

The above-mentioned alkylated derivatives can be prepared by reacting the kalamycin derivative compounds of Formulas (III) and (V-wherein R is hydrogen) with an alkyl halide, preferably an alkyl iodide, in the presence of a halide precipitating agent, for example, silver oxide, silver carbonate, and the like. The reaction, advantageously, can be conducted at reflux for a period of from 4-10 hours. Lower reaction temperatures, i.e., less than reflux, also can be used but these tend to prolong the reaction time unnecessarily. The course of the reaction can be followed by thin-layer chromatography (TLC) using silica gel which can be developed by a solvent system consisting of ethyl acetate-cyclohexane.

Examples of alkyl halides which can be used in the process are methyl iodide, ethyl iodide, ethyl bromide, ethyl chloride, propyl iodide, butyl iodide, pentyl iodide, hexyl iodide, heptyl iodide, octyl iodide, nonyl iodide, decyl iodide, undecyl iodide, dodecyl iodide, tridecyl iodide, tetradecyl iodide, pentadecyl iodide, hexadecyl iodide, and isomers thereof, and the like.

Acylates of kalamycin derivative compounds of Formulas (III), (Vwherein R is hydrogen), and (VI- wherein R is hydrogen), are obtained by direct acylation of the kalamycin derivative compound with an acid halide or an anhydride of a selected hydrocarbon carboxylic acid in the presence of an acid-binding agent, for example, a tertiary amine. Suitable tertiary amines include heterocyclic amines such as pyridine, quinoline, and isoquinoline; trialkylamines such as trimethylamine, triethylamine, triisopropylamine, and the like;'N,N-dialkylanilines such as dimethylaniline, diethylaniline, and the like; and N- alkylpiperidines such as N-ethylpiperidine, N-methylpiperidine, and the like. The preferred base is pyridine. Carboxylic acids suitable for esterification include (a) saturated or unsaturated, straight or branched chain aliphatic carboxylic acids, for example, acetic, propionic, butyric, isobutyric, tert-butyl-acetic, valeric, isovaleric, caproic, caprylic, decanoic, dodecanoic, lauric, tridecanoic, myristic, pentadecanoic, palmitic, margaric, stearic, acrylic, crotonic, undecylenic, oleic, hexynoic, heptynoic, octynoic acids, and the like; (b) saturated or unsaturated, alicyclic carboxylic acids, for example, cyclobutanecarboxylic acid, cyclopentanecarboxylic acid, cyclopentenecarboxylic acid, methylcyclopentenecarboxylic acid, cyclohexanecarboxylic acid, dimethylcyclohexenecarboxylic acid, dipropylcyclohexanecarboxylic acid, and the like; saturated or unsaturated, alicyclic aliphatic carboxylic acids, for example, cyclopentaneacetic acid, cyclopentanepropionic acid, cyclopenteneacetic acid, cyclohexanebutyric acid,

methylcyclohexaneacetic acid, and the like; (d) aromatic carboxylic acids, for example, benzoic acid, toluic acid, naphthoic acid, ethylbenzoic acid, isobutylbenzoic acid, methylbutylbenzoic acid, and the like; and (e) aromaticaliphatic carboxylic acids, for example, phenylacetic acid, phenylpropionic acid, phenylvaleric acid, cinnamic acid, phenylpropiolic acid and naphthylacetic acid, and the like. Suitable halo-, nitro-, hydroxy-, amino-, cyano-, thiocya- 110-, and lower alkoxyhydrocarbon carboxylic acids include hydrocarbon carboxylic acids as given above which are substituted by one or more of halogen, nitro, hydroxy, amino, cyano, or thiocyano, or loweralkoxy, advantageously loweralkoxy of not more than six carbon atoms, for example, methoxy, ethoxy, propoxy, butoxy, amyloxy, hexyloxy, and isomeric forms thereof. Examples of such substituted hydrocarbon carboxylic acids are mono-, di-, and trichloroacetic acid; ocand fi-chloropropionic acid; a and 'y-bromobutyric acid; aand n-iodovaleric acid; mevalonic acid; 2- and 4-chlorocyclohexanecarboxylic acid; shikimic acid; Z-nitro-l-methylcyclobutanecarboxylic acid; l,2,3,4,5,6-hexachlorocyclohexanecarboxylic acid; 3-bromo-2-methylcyclohexanecarboxylic acid; 4- and 5- bromo-Z-methylcyclolrexanecarboxylic acid; 5- and 6-bromo 2 methylcyclo-hexanecarboxylic acid; 2,3-dibromo- 2-methylcyclohexanecarboxylic acid; 2,5-dibromo-2-methylcyclohexanecarboxylic acid; 4,5 dibromo Z-methylcyclohexanecarboxylic acid; 5,6-dibromo-Z-methylcyclohexanecarboxylic acid; 3-brorno-3-methylcyclohexanecarboxylic acid; 6-bromo-3-methylcyclohexanecarboxylic acid; 1,6 dibromo 3-methylcyclohexanecarboxylic acid; 2- bromo 4 methylcyclohexanecarboxylic acid; 1,2-dibromo 4-methylcyclohexanecarboxylic acid; 3-brorno- 2,2,3-trirnethylcyclopentanecarboxylic acid; 1-brom0-3,5- dimethylcyclohexanecarboxylic acid; homogentisic acid, 0-, m-, and p-chlorobenzoic acid; anisic acid; salicylic acid; p-hydroxybenzoic acid; fl-resorcylic acid; gallic acid; veratric acid; trirnethoxybenzoic acid; trimetlroxycinnamic acid; 4,4'-dichlorobenzilic acid; o-, m-, and p-nitrobenzoic acid; cyanoacetic acid; 3,4- and 3,5-dinitrobenzoic acid; 2,4,6-trinitrobenzotic acid; thiocyanoacetic acid; cyanopropionic acid; lactic acid; ethoxyforrnic acid (ethyl hydrogen carbonate); and the like.

The acylation, advantageously, is conducted by treating a suspension of the kalamycin derivative compound in a tertiary amine with an acid halide or anhydride and heating the resulting mixture, if desired, for a short period at about 80 C. to complete the reaction. Water can be added to the reaction mixture to hydrolyze the acylating agent, and the desired product can be isolated by conventional procedures.

Salts of kalamycin degradation products and derivatives can be made whenever the hydroxyl group on the 9 position of the molecule, or the carboxyl group, as in kalamycinic acid (II) and dehydrokalamycinic acid (V wherein R and R are hydrogen), is available to maintain the acidic nature of the compound. Salts are formed by employing the free acid of the kalamycin degradation product or derivative thereof, and an inorganic or organic base. These salts can be prepared, as for example, by dissolving the free acid compound in water, adding a dilute base until the pH of the solution is about 10.0 to 11.0, and freeze-drying the solution to provide a dried residue consisting of the desired salt. Salts which can be formed include the sodium, potassium, and calcium salts. Other salts, including those with organic bases such as primary, secondary, and tertiary monoamines as well as with polyamine, also can be formed using the above-described or other commonly employed proce dures. Other valuable salts are obtained with therapeutically eifective bases which impart additional therapeutic effects thereto. Such bases are, for example, the purine bases such as theophyllin, theobromin, caffeine, or derivatives of such purine bases; antihistaminic bases which are capable of forming salts with weak acids; pyridine compounds such as nicotinic acid amide, isonicotinic acid hydrazide, and the like; phenylalkylamines such as adrenaline, ephedrine, and the like; choline, and others. Salts of kalamycin degradation products and derivatives can be used for the same biological purposes as the free acid.

The compounds of the invention show antifungal activity against various fungi. For example, kalamycinic acid (II) and ethyl dehydrokalamycinate (V-wherein R is hydrogen and R is ethyl) are active against Blast0 myces dermazitidis, Hormodendrum compactum, Trichopltyton rubrum, Trichophyton asteroides, and T richophyton mentagrophytes; whereas ethyl kalamycinate (III-- wherein R is ethyl) is active against Nocardia asteroides, Blastomyces dermatitidis, Coccidz'oides immitis, Hormodendrum compaczum, Cryptococcus neoformans (kalamycinic acid also is active against this fungus), H istoplasma capsulatum, and Monosporz'um apiospermum. Thus, the novel compounds of the invention can be used as antifungal agents in various environments to inhibit the growth of the above-mentioned fungi. For example, kalamycinic acid and ethyl dehydrokalamycinate can be used to inhibit the growth of Cryptococcus neoformans which is found in pigeon roosts. The novel compounds of the invention can also be used as antifungal agents in the shoe uppers disclosed in US. Pat. 3,130,505. Furthermore, the novel compounds of the invention can be used to swab laboratory benches and equipment in a mycological laboratory. The acylated compounds of the invention can be used to upgrade crude preparations of the unacylated compound. For example, crude kalamycinic acid (II) can be acylated, as disclosed hereinafter, to remove unacylatable impurities. The acylated kalamycinic acid (11) then can be deacylated by mild acid hydrolysis, for example, 1 N HCl, to provide a purified preparation of kalamycinic acid.

The following examples are illustrative of the process and products of the present invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Kalamycinic acid 3,4,5,10-tetrahydro 4,9 dihydroxy-1-methyl-5,10-dioxo-lH-naphtho[2,3-c1pyran-3-acetic acid A one-gram (3.33 mmole) quantity of kalamycin was dissolved with stirring in 10 ml. of 1 N sodium hydroxide. When all of the kalamycin was dissolved, the solution was neutralized by the addition of 11 ml. of 1 N hydro- EXAMPLE 2 Ethyl kalamycinate 3,4,5,10-tetrahydro 4,9 dihydroxy-1-methyl-5,l0-dioxo-1H-naphtho[2,3-c] pyran-3-acetic acid, ethyl ester 1.5 Hours H O C H O H O C H: I II I l Alcoholic 0 A Q-cmfimcnn ll 0 O O H A solution was prepared containing 2 g. (6.67 mmole) of kalamycin in 300 ml. of ethanolic hydrogen chloride (2.0 N). After 1.5 hrs., the solution was evaporated to dryness on a rotary evaporator. The resulting residue was dried in a desiccator under high vacuum. This material was then chromatographed on a 1.75-diameter column containing 250 g. of silica gel which was developed by a solvent system consisting of ethyl acetate-cyclohexane (3:5). Fractions of 20 ml. were collected. Fractions from tubes 35 through 42 were combined, evaporated to dryness in the desiccator; yield, 300 mg. This material was crystallized from chloroform and Skellysolve B; yield, 300 mg. of ethyl kalamycinate having a melting point of 127 C.

Elemental Analysis.Calcd for C H O (molecular weight=346.2) (percent): C, 62.42; H, 5.24; O, 32.34. Found (percent): C, 6 1.96, 61.99; H, 5.20, 5.17.

EXAMPLE 3 Ethyl dehydrokalamycinate 404,110,100: tetrahydro 9 hydroxy-1-methyl-5,10- dioxo 1H naphtho[2,3-c]pyran-3-acetic acid, ethyl ester 0 H (H) 0 Ha H (I) (u) (313;

A Alcohol A d l 2 hour VV-CHz-[Cf-O C 2116 II I ll 0 of silica gel developed with a solvent system consisting of ethyl acetate-cyclohexane (1:4). Fractions of 10 ml.

were collected. A pool of fractions from tubes 30-55 was evaporated to dryness and then crystallized from hot Skellysolve B; yield, 300 mg. of ethyl dehydrokalamycinate having a melting point of 104 C.

' 8 Elemental Analysis.-Calcd for C H O (molecular weight=328.31) (percent): C, 65.8; H, 4.9; O, 29.2. Found (percent): C, 65.45; H, 5.05.

Treatment of ethyl dehydrokalamycinate with 1 N ethanolic (50% aqueous) potassium hydroxide for 1 hr.

at room temperature, followed by acidification, produces dehydrokalamycinic acid.

EXAMPLE 4 Conversion of kalamycinic acid to kalamycin A solution of 1 g. (3.1 mm.) of kalamycinic acid, as prepared in Example 1, in 150 ml. of ethanolic hydrogen chloride was maintained at room temperature for 20 min. This solution was then evaporated to dryness in vacuo and chromatographed in a 1.25"-column over 150 g. of silica gel (Darmstadt). The column was developed with a solvent system consisting of ethyl acetate and cyclohex- 'ane (3:5). Of the ltl-mhfractions collected, tubes through 100, when pooled, contained 370 mg. of kalamycin.

EXAMPLE 5 By substituting the ethanolic hydrogen chloride in Example 2 by methanolic hydrogen chloride, propanolic hydrogen bromide, butanolic hydrogen chloride, pentanolic hydrogen sulfate, hexanolic hydrogen phosphate, heptanolic hydrogen chloride, octanolic hydrogen chloride, nonanolic hydrogen chloride, decanolic hydrogen chloride, undecanolic hydrogen chloride, dodecanolic hydrogen chloride, tridecanolic hydrogen chloride, tetradecanolic hydrogen chloride, pentadecanolic hydrogen bromide and hexadecanolic hydrogen sulfate there is obtained methyl-, propyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, and hexadecyl kalamycinate, respectively.

EXAMPLE 6 By substituting the ethanolic hydrogen chloride in Example 3 by methanolic hydrogen chloride, propanolic hydrogen bromide, butanolic hydrogen chloride, pentanolic hydrogen sulfate, hexanolic hydrogen phosphate, heptanolic hydrogen chloride, octanolic hydrogen chloride, nonanolic hydrogen chloride, decanolic hydrogen chloride, undecanolic hydrogen chloride, dodecanolic hydrogen chloride, tridecanolic hydrogen chloride, tetradecanolic hydrogen chloride, pentadecanolic hydrogen bromide and hexadecanolic hydrogen sulfate there is obtained methyl-, propyl-, butyl-, pentyl-, hexy1-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl,- tetradecyl-, pentadecyl-, and hexadecyl dehydrokalamycinate, respectively.

EXAMPLE 7 To 3.2 g. of kalamycinic acid in ml. of dry acetone is added 300 ml. of methyl iodide. This solution is brought to reflux and 1.5 g. of silver oxide is' added. A second 1.5 g. portion of silver oxide is added 1 hr..later, and a third 2 hrs. after the initial addition. Reflux is continued with stirring and the course of the reaction is followed on thin-layer chromatography using silica gel developed by ethyl acetate-cyclohexane (3:5 Upon disappearance of the starting material, the mixture is filtered and the mixture is acidified with 0.1 N HCl. The filtrate is then evaporated to dryness and redissolved in ethanolic potassium hydroxide (1 N). This is acidified with ethanolic hydrogen chloride after 2 hrs. and evaporated to dryness. The residue is dissolved in boiling acetoneand evaporated to dryness. Purification is accomplished by silica gel chromatography developed by ethyl acetate and cyclohexane, to yield 4,9-di-O-methyl-kalamycinic acid.

EXAMPLE 8 v By substituting the methyl iodide in Example 7 by ethyl iodide, propyl iodide, butyl iodide, pentyl iodide, hexyl iodide, heptyl iodide, octyl iodide, nonyl iodide, decyl iodide, undecyl iodide, dodecyl iodide, tridecyl u iodide, tetradecyl iodide, pentadecyl iodide, and hexadecyl iodide, there is obtained a corresponding 4,9-di-O-alkylkalamycinic acid.

EXAMPLE 9 EXAMPLE 10 By substituting the acetic anhydride in Example 9 by anhydrides of hydrocarbon carboxylic acids of from 3 to 12 carbon atoms, inclusive, there is obtained the corresponding 4,9-di-O-acyl-kalamycinic acid.

EXAMPLE 11 By substituting the acetic anhydride in Example 9 by anhydrides of hydrocarbon carboxylic acids of from 10 to 18 carbon atoms, inclusive, in the presence of a solvent, for example, diethyl ether, methylene chloride, carbon tetrachloride, and the like, there is obtained the corresponding 4,9-di-O-acyl-kalamycinic acid.

EXAMPLE 12 To 3.3 g. of ethyl dehydrokalamycinate, the compound produced in Example 3, in 150 ml. of dry acetone is added 150 ml. of methyl iodide. This solution is brought to reflux and 1.5 g. of silver oxide is added. A second 1.5 g. portion of silver oxide is added 1 hr. later, and a third 2 hrs. after the initial addition. Reflux is continued with stirring and the course of the reaction is followed on thin-layer chromatography using silica gel developed by ethyl acetate-cyclohexane. Upon disappearance of the starting material, the mixture is filtered and the mixture acidified with 0.1 N HCl. The filtrate is then evaporated to dryness and dried in a desiccator. The residue is dissolved in boiling acetone and purification of ethyl 9-O-methyl-dehydrokalamycinate is then accomplished by chromatography.

EXAMPLE 13 By substituting the methyl iodide in Example 12 by ethyl iodide, propyl iodide, butyl iodide, pentyl iodide, hexyl iodide, heptyl iodide, octyl iodide, nonyl iodide, decyl iodide, undecyl iodide, dodecyl iodide, tridecyl iodide, tetradecyl iodide, pentadecyl iodide, and hexadecyl iodide there is obtained a corresponding ethyl 9-O-alkyl-dehydrokalamycinate.

EXAMPLE 14 A sample of ethyl dehydrokalamycinate (approximately 45 mg.), as produced in Example 3, is mixed with acetic anhydride (2 ml.) and 1 drop of pyridine is added. The mixture is heated on the steam bath for /2 hr., then stored at room temperature 1 day. Thereupon it is added to ice-water and a solid recovered. This solid is purified by chromatography on silica gel, developed with a solution of ethyl acetate and cyclohexane, to yield ethyl 9-O-acetyldehydrokalamycinate.

EXAMPLE 15 By substituting the acetic anhydride in Example 14 by anhydrides of hydrocarbon carboxylic acids of from 3 to 12 carbon atoms, inclusive, there is obtained the corresponding ethyl 9-O-acyl-dehydrokalamycinate.

EXAMPLE 16 By substituting the acetic anhydride in Example 14 by anhydrides of hydrocarbon carboxylic acids of from 10 to 18 carbon atoms, inclusive, in the presence of a solvent, for example, diethyl ether, methylene chloride, carbon tetrachloride, and the like, there is obtained the corresponding ethyl 9-O-acyl-dehydrokalamycinate.

EXAMPLE 17 By substituting the ethyl dehydrokalamycinate in Ex- 2 amples 12, and 13, by the alkyl dehydrokalamycinates,

as prepared in Example 6, there are obtained the corresponding 9-O-alkyl derivatives of such alkyl dehydrokalamycinates.

EXAMPLE 18 Ethyl 4-deoxy-4-chloro-kalamycinate 4-chloro-3,4,5,10 tetrahydro 9 hydroxy 1 methyl- 5,10-dioxo-1H-naphtho[2,3-c]pyran-B-acetic acid.

1 W EtOH V E01 V\ 2.5 Hours 1 l 0 a O O A 5 g. quantity of kalamycin was dissolved in 500 ml. of ethanolic hydrogen chloride (2 N) for about 3.5 hrs. at room temperature. The solution was evaporated to dryness on the rotary evaporator. The resulting residue was further evaporated to dryness and chromatographed on a column (1.5" in diameter) containing 300 g. of silica gel, developed with a solution of ethyl acetate and cyclohexane in the ratio of 3:5. Ten ml. fractions were collected. Fractions 10 to 34 were combined, evaporated and crystallized from ml. of a mixture of chloroform and Skellysolve B (approximately 1:20); yield, 365 mg. of orange crystals of ethyl 4-deoxy-4-chloro-kalamycinate having a melting point of -133 C.

Elemental analysis.-Calcd for C H O Cl (molecular weight:364) (percent): C, 59.26; H, 4.70; O, 26.32; Cl, 9.72. Molecular Weight-364 (by mass spectrum dc termination) EXAMPLE 20 By substituting the ethanolic hydrogen chloride in Example 19 by ethanolic hydrogen bromide, there is obtained 4-deoxy-4-bromo-kalamycinic acid, ethyl ester.

EXAMPLE 21 By substituting the ethanolic hydrogen chloride in EX- ample 19 by methanolic hydrogen chloride and methanolic hydrogen bromide, there is obtained methyl 4-deoxy- 4-chloro-kalarnycinate and methyl 4-deoxy-4-bromo-kalamycinate, respectively.

EXAMPLE 22 A sample of ethyl 4-deoxy-4-chloro-kalamycinate, as prepared in Example 19 (approximately 50 mg.), is mixed with acetic anhydride (2 ml.) and 1 drop of pyridine is added. The mixture is heated on the steam bath for /2 hr., then stored at room temperature for 1 day. Thereupon it is added to ice-water and a solid recovered. This solid is purified by chromatography on silica gel, developed with a solution of ethyl acetate and cyclohexane, to yield ethyl 9-O-acetyl-4-deoxy-4-chlorokalamycinate.

EXAMPLE 23 By substituting the acetic anhydride in Example 22 by anhydrides of hydrocarbon carboxylic acids of from 3 to 12 carbon atoms, inclusive, there is obtained the corresponding ethyl 9-O-acyl-4-deoxy-4-chloro-kalamycinate.

EXAMPLE 24 By substituting the acetic anhydride in Example 22 by anhydrides of hydrocarbon carboxylic acids of from 10 to 18 carbon atoms, inclusive, in the presence of a solvent, for example, diethyl ether, methylene chloride, carbon tetrachloride, and the like, there is obtained the corresponding ethyl 9 O acyl-4-deoxy-4-chloro-kalamy: cinate.

EXAMPLE 25 By substituting the ethanolic hydrogen chloride in Example 19' by methanolic hydrogen chloride there is obtained methyl 4-deoxy-4-chloro-kalamycinate.

EXAMPLE 26 By substituting the ethanolic hydrohalic acids in Examples 20 and 21 by methanolic solutions of the same hydrohalic acids there is obtained the corresponding methyl 4-deoxy-4-halo-kalamycinates.

EXAMPLE 27 Acylation of dehydrokalamycinic acid A sample of dehydrokalamycinic acid (approximately 40 mg.) is mixed with acetic anhydride (4 ml.) and 2 drops of pyridine are added. The mixture is heated on the steam bath for /2 hr., then stored at room temperature 1 day. Thereupon it is added to ice-water and a solid material recovered. This solid is purified by chromatography on silica gel, developed with a solution of ethyl acetate and cyclohexane to yield 4,9-di-O-acetyl-dehydrokalamycinic acid.

EXAMPLE 28 By substituing the acetic anhydride in Example 27 by anhydrides of hydrocarbon carboxylic acids of from 3 to 12, carbon atoms, inclusive, there is obtained the corresponding 4,9-di-O-acyl-dehydrokalamycinic acid.

EXAMPLE 29 By substituting the acetic anhydride in Example 27 by anhydrides of hydrocarbon carboxylic acids of from 10 to 18 carbon atoms, inclusive, in the presence of a solvent, for example, diethyl ether, methylene chloride, carbon tetrachloride, and the like, there is obtained the corresponding 4,9-di-O-acyl-dehydrokalamycinic acid.

I claim:

1. A compound of the formula:

wherein R is hydrogen or alkyl of from 1 to 16 carbon atoms, inclusive, and the isomeric forms thereof.

2. Kalamycin acid, a compound according to claim 1, wherein R is hydrogen.

3. Ethyl kalamycinate, a compound according to claim 1, wherein R is ethyl.

4. A compound of the formula:

.- 0 R IV wherein R is hydrogen or alkyl of from 1 to 16 carbon atoms, inclusive, and the isomeric forms thereof; wherein R is alkyl of from 1 to 16 carbon atoms, inclusive, and the isomeric forms thereof, or hydrocarbon carboxylic acid acyl of from 2 to 18 carbon atoms, inclusive; halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and loweralkoxy-substituted hydrocarbon carboxylic acid acyl of from 2 to 18 carbon atoms, inclusive; and lower-alkoxycarbonyl.

5. A compound of the formula:

wherein R is hydrogen or alkyl of from 1 to 16 carbon atoms, inclusive, and isomeric forms thereof; R is hydrogen, or alkyl of from 1 to 16 carbon atoms, inclusive, and isomeric forms thereof, or hydrocarbon carboxylic acid acyl of from 2 to 18 carbon atoms, inclusive; halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower-alkoxysubstituted hydrocarbon carboxylic acid acyl of from 2 to- 18 carbon atoms, inclusive; and lower-alkoxycarbonyl. 6. Ethyl dehydrokalamycinate, a compound according to claim 5 wherein R is ethyl and R is hydrogen.

7. A compound of the formula:

wherein -R is alkyl of from 1 to 16 carbon atoms, inclusive, and isomeric forms thereof; R' is hydrogen or hydrocarbon carboxylic acid acyl of from 2 to 18 carbon atoms, inclusive; halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower-alkoxy-substituted hydrocarbon ca'rboxylic acid acyl of from 2 to 18 carbon atoms, inclusive,

and loweralkoxycarbonyl; and X is halo.

8. Ethyl 4-deoxy-4-chloro-kalamycinate, a compound according to claim 7, wherein R is ethyl, R is hydrogen, and X is chloro.

9. A process for preparing a compound as defined in claim 1 wherein R is hydrogen, which comprises reacting a compound of the formula:

sodium hydroxide with kalamycin and acidifying the resulting reaction mixture to produce kalamycinic acid.

11. A process for preparing a compound as defined in claim 1 wherein R is alkyl of from 1 to 16 carbonatoms, inclusive, and isomeric forms thereof, which comprises reacting a compound of the formula:

with an alcohol, in the presence of a mineral acid, for a time sufficient to open the lactone ring and form the ester compound, neutralizing the reaction mixture, and isolating the desired compound.

12. A process, according to claim 11, for preparing a compound of the formula:

which comprises reacting kalarnycin with 2 N ethanolic hydrogen chloride for about 1.5 hours at room temperature, neutralizing the reaction mixture with a mineral base, and isolating the desired compound.

13. A process for preparing a compound of the formula:

HO O OH:

wherein R is alkyl of from 1 to 16 carbon atoms, inclusive, and isomeric forms thereof; and X is halo, which comprises reacting a compound of the formula:

with an alcohol of from 1 to 16 carbon atoms, inclusive, and isomeric forms thereof, in the presence of a hydrohalic acid for about 2.5 hours, and isolating the compound so produced.

14. A process, according to claim 13, for preparing a compound of the formula:

HO O I ll References Cited UNITED STATES PATENTS 2,852,530 9/1958 Ford 260-343.6 2,960,441 11/1960 van Wessem 260343.6 XR 3,007,940 11/1961 Shavel et al. 260-3436 3,246,014 4/1966 Jung et a]. 260345.2 X=R 3,300,382 1/196'7 Bergy et a1. 260-343.6

HENRY R. JILES, Primary Examiner J. M. FORD, Assistant Examiner U .8. Cl. X. R, 260-999 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent; No. 3,524,868 Dated August 18, 1970 Inventor) Herman Hoeksema It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

CoI umn 3, I ine 38, for "aqu I I Ibr I um" read equ II ibr i um I ine 42, for "decaoI read decanoI I Ine 47, for "betwn" read between Column 11, I ine 58, for

"Kalam cIn" read KaIamycInIc CoI umn 12, I Ine 13,

for "R read R' I ine 28, for -CH -C0" read CH2-C-0R SIGNED AND SEALED was 1971 fi Arum:

1: H" m Gomissiow of Patent) 001 umn 2, I ine 59, for CH -C=0R" read --CH -C-OR FORM P0-1050 (10-69) USCOMM-DC scan-Pea LII-S. GOVERNMENT PRINTING OFFICE: III! 0-38-38 

